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1.
Mol Biol (Mosk) ; 53(4): 685-691, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397442

RESUMO

In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Proteoma/análise , Proteômica/métodos , Coloração e Rotulagem/métodos , Humanos , Focalização Isoelétrica , Proteoma/química , Espectrometria de Massas em Tandem
2.
J Clin Pathol ; 72(11): 755-761, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31256009

RESUMO

AIMS: Morphological differentiation among different blast cell lineages is a difficult task and there is a lack of automated analysers able to recognise these abnormal cells. This study aims to develop a machine learning approach to predict the diagnosis of acute leukaemia using peripheral blood (PB) images. METHODS: A set of 442 smears was analysed from 206 patients. It was split into a training set with 75% of these smears and a testing set with the remaining 25%. Colour clustering and mathematical morphology were used to segment cell images, which allowed the extraction of 2,867 geometric, colour and texture features. Several classification techniques were studied to obtain the most accurate classification method. Afterwards, the classifier was assessed with the images of the testing set. The final strategy was to predict the patient's diagnosis using the PB smear, and the final assessment was done with the cell images of the smears of the testing set. RESULTS: The highest classification accuracy was achieved with the selection of 700 features with linear discriminant analysis. The overall classification accuracy for the six groups of cell types was 85.8%, while the overall classification accuracy for individual smears was 94% as compared with the true confirmed diagnosis. CONCLUSIONS: The proposed method achieves a high diagnostic precision in the recognition of different types of blast cells among other mononuclear cells circulating in blood. It is the first encouraging step towards the idea of being a diagnostic support tool in the future.


Assuntos
Interpretação de Imagem Assistida por Computador/métodos , Leucemia/patologia , Leucócitos/patologia , Aprendizado de Máquina , Reconhecimento Automatizado de Padrão/métodos , Coloração e Rotulagem/métodos , Doença Aguda , Coleta de Amostras Sanguíneas , Linhagem da Célula , Diagnóstico Diferencial , Humanos , Leucemia/sangue , Leucemia/classificação , Valor Preditivo dos Testes , Reprodutibilidade dos Testes
3.
Plant Dis ; 103(9): 2330-2336, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31298992

RESUMO

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.


Assuntos
Azul Evans , Plasmodioforídeos , Esporos de Protozoários , Coloração e Rotulagem , Azul Evans/metabolismo , Doenças das Plantas , Plasmodioforídeos/fisiologia , Esporos de Protozoários/fisiologia , Coloração e Rotulagem/métodos , Coloração e Rotulagem/normas
4.
Virchows Arch ; 475(2): 191-199, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31264038

RESUMO

Pre-analytical factors, such as fixation time, influence morphology of diagnostic and predictive immunohistochemical staining, which are increasingly used in the evaluation of lung cancer. Our aim was to investigate if variations in fixation time influence the outcome of immunohistochemical staining in lung cancer. From lung resections, specimen with tumor size bigger than 4 cm, 10 samples were obtained: 2 were put through the standard fixation protocol, 5 through the delayed, and 3 through the prolonged fixation protocol. After paraffin embedding, tissue microarrays (TMAs) were made. They were stained with 20 antibodies and scored for quality and intensity of staining. Samples with delay in fixation showed loss of TMA cores on glass slides and deterioration of tissue quality leading to reduction in the expression of CK 7, Keratin MNF116, CAM 5.2, CK 5/6, TTF-1, C-MET, Napsin A, D2-40, and PD-L1. Prolonged fixation had no influence on the performance of immunohistochemical stains. Delay of fixation negatively affects the expression of different immunohistochemical markers, influencing diagnostic (cytokeratins) and predictive (PD-L1) testing. These results emphasize the need for adequate fixation of resection specimen.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , Imuno-Histoquímica/métodos , Neoplasias Pulmonares/patologia , Fixação de Tecidos/métodos , Humanos , Coloração e Rotulagem/métodos
5.
Nat Commun ; 10(1): 2539, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182711

RESUMO

Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, 19F NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed.


Assuntos
Glicina/química , Insulina/química , Proteínas/química , Coloração e Rotulagem/métodos , Aldeídos/química , Escherichia coli , Corantes Fluorescentes/química , Flúor , Células HEK293 , Humanos , Receptor de Insulina/química
6.
Eur J Histochem ; 63(2)2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31232013

RESUMO

Mucins are of great interest in intestinal research and histochemical methods are often employed to identify them. Since it is in the nature of mucins that they are "hard to hold onto" once they come into contact with water, a frequently used medium in histochemistry, there are a number of challenges that may decrease diagnostic accuracy. As the outcome of methods published for microscopic detection of mucosubstances proved to be unsatisfactory in our hands, the aim was the establishment of a reliable and reproducible protocol. Tissue samples were available from pig feeding experiments. In the present study, we focus on a fixation / staining procedure without making comparisons between differently fed pigs. Several fixation and staining procedures were evaluated for their use in semiautomatic quantification and quality assessment of different mucus fractions simultaneous on one tissue section. Cryostat sectioning, subsequent fixation steps with heat, ethanol and modified Bouin's solution, followed by triple staining with high iron diamine, alcian blue and periodic acid-Schiff turned out to be the best method to identify sulfomucin, sialomucin and neutral mucin simultaneous on one tissue section. This methodology resulted in very good morphology of goblet cells with intact mucin containing vesicles within the cells, which was comparable to ultrastructural electron microscopical observations. Semiautomatic quantification of different mucins was possible. In conclusion, reliable mucus quantification and assessment of mucus quality requires strictly tested procedures. According to our experience, the most important aim after cryosectioning is fast fixation of the mucosubstances, which requires a combination of different fixation steps.


Assuntos
Trato Gastrointestinal/química , Histocitoquímica/métodos , Mucinas/análise , Coloração e Rotulagem/métodos , Animais , Vesículas Citoplasmáticas/química , Trato Gastrointestinal/citologia , Células Caliciformes/química , Células Caliciformes/citologia , Mucinas/química , Mucinas/classificação , Suínos
7.
Nat Commun ; 10(1): 2770, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235780

RESUMO

The ability to link soil microbial diversity to soil processes requires technologies that differentiate active microbes from extracellular DNA and dormant cells. Here, we use BONCAT (bioorthogonal non-canonical amino acid tagging) to measure translationally active cells in soils. We compare the active population of two soil depths from Oak Ridge (Tennessee, USA) and find that a maximum of 25-70% of the extractable cells are active. Analysis of 16S rRNA sequences from BONCAT-positive cells recovered by fluorescence-activated cell sorting (FACS) reveals that the phylogenetic composition of the active fraction is distinct from the total population of extractable cells. Some members of the community are found to be active at both depths independently of their abundance rank, suggesting that the incubation conditions favor the activity of similar organisms. We conclude that BONCAT-FACS is effective for interrogating the active fraction of soil microbiomes in situ and provides a new approach for uncovering the links between soil processes and specific microbial groups.


Assuntos
Bactérias/isolamento & purificação , Citometria de Fluxo/métodos , Microbiota , Microbiologia do Solo , Aminoácidos/análise , Aminoácidos/química , Bactérias/genética , Bactérias/metabolismo , Corantes Fluorescentes/química , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Coloração e Rotulagem/métodos
8.
Acta Cytol ; 63(5): 401-410, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31112943

RESUMO

BACKGROUND: Oral exfoliative cytology is a noninvasive and nonpainful technique for early diagnosis of oral potentially malignant disorders and oral cancer, and the use of cytomorphometry ameliorates its diagnostic reliability. The objective of the present study was to analyze methyl green-pyronin Y (MGP)-stained oral exfoliated cells (OECs) of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) by cytomorphometry. MATERIALS AND METHOD: An observational study was conducted on 150 individuals equally divided into three groups: normal mucosa, OL, and OSCC. Smears were prepared from OECs and stained with MGP. Cytomorphometry was done for 100 cells per subject, and various cell and nuclear parameters were measured and calculated. RESULTS: The Kruskal-Wallis test with post hoc correlation showed significant differences in nucleus and cell diameter (ND, CD), nucleus and cell area (NA, CA), nucleus and cell perimeter (NP, CP), and nucleus to cytoplasmic (N:C) ratio for diameter, perimeter, and area. Spearman's ρ correlation of various N:C ratio methods showed good correlation between N:C perimeter and diameter ratio, N:C diameter and ellipse ratio, and N:C area and ellipse ratio. Additional morphological factors showed significant relations for both cell and nuclear regularity factor, shape factor, and nuclear contour index. DISCUSSION: MGP-based cytomorphometry showed a significant decrease in CD, CA, and CP and increase in ND, NA, NP, and N:C ratio from normal mucosa to OL and OSCC. MGP proved its worth as an effective stain for OECs, despite its strict standardization.


Assuntos
Corantes/química , Citodiagnóstico/métodos , Detecção Precoce de Câncer/métodos , Leucoplasia Oral/patologia , Verde de Metila/química , Neoplasias Bucais/patologia , Pironina/química , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Coloração e Rotulagem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , não Fumantes , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fumantes , Manejo de Espécimes
9.
Appl Opt ; 58(12): 3104-3114, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31044784

RESUMO

Modern microscopes are designed with functionalities that are tailored to enhance image contrast. Dark-field imaging, phase contrast, differential interference contrast, and other optical techniques enable biological cells and other phase-only objects to be visualized. Quantitative phase imaging refers to an emerging set of techniques that allow for the complex transmission function of the sample to be measured. With this quantitative phase image available, any optical technique can then be simulated; it is trivial to generate a phase contrast image or a differential interference contrast image. Rheinberg illumination, proposed almost a century ago, is an optical technique that applies color contrast to images of phase-only objects by introducing a type of optical staining via an amplitude filter placed in the illumination path that consists of two or more colors. In this paper, the complete theory of Rheinberg illumination is derived, from which an algorithm is proposed that can digitally simulate the technique. Results are shown for a number of quantitative phase images of diatom cells obtained via digital holographic microscopy. The results clearly demonstrate the potential of the technique for label-free color staining of subcellular features.


Assuntos
Diatomáceas/citologia , Holografia/métodos , Iluminação , Microscopia de Contraste de Fase/métodos , Coloração e Rotulagem/métodos , Algoritmos
10.
J Enzyme Inhib Med Chem ; 34(1): 946-954, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31039618

RESUMO

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASLtag). The anchored chimeric H5-SspCA was active for the CO2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASLtag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions.


Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Bactérias Gram-Negativas Quimiolitotróficas/enzimologia , Coloração e Rotulagem/métodos , Temperatura Ambiente , Estabilidade Enzimática , Modelos Moleculares , Relação Estrutura-Atividade
11.
Nat Commun ; 10(1): 1930, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036827

RESUMO

Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.


Assuntos
Cromatina/química , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Cromatina/metabolismo , Regulação da Expressão Gênica , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Código das Histonas , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transposases/genética , Transposases/metabolismo
12.
Nat Protoc ; 14(6): 1708-1733, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31028373

RESUMO

Understanding the structure-function relationships between diverse cell types in a complex organ environment requires detailed in situ reconstruction of cell-associated molecular properties in the context of 3D, macro-scale tissue architecture. We recently developed clearing-enhanced 3D (Ce3D), a simple and effective method for tissue clearing that achieves excellent transparency; preserves cell morphology, tissue architecture, and reporter molecule fluorescence; and is robustly compatible with direct immunolabeling. These characteristics permit high-quality multiplex fluorescence microscopy of large tissue volumes, as well as image analysis using advanced platforms such as volumetric histocytometry, collectively allowing quantitative characterization of cells with respect to their spatial positioning within tissues on the basis of phenotypic and functional markers. Ce3D clearing is fast, achieving robust transparency of most tissues within 24 h, albeit still necessitating additional time for staining, imaging, and analysis (1-2 weeks). Here, we provide detailed procedures for tissue clearing using Ce3D, including optimized workflows for tissue processing and staining, as well as treatment of difficult-to-clear organs such as the brain. We also describe a new procedure for RNA detection in Ce3D-treated tissues, as well as provide additional details for the volumetric histocytometry data processing steps. Finally, we discuss limitations and work-around strategies for improving antibody-based tissue immunolabeling, fluorophore multiplexing, large-volume microscopy, and computational analysis of large image datasets. Together, these detailed procedures and solutions for high-resolution volumetric microscopy with Ce3D enable quantitative visualization of cells and tissues at a high level of detail, allowing exploration of cellular spatial relationships in a variety of tissue settings.


Assuntos
Imagem Tridimensional/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Animais , Química Encefálica , Feminino , Imuno-Histoquímica/métodos , Masculino , Camundongos , RNA/análise , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos
13.
Nat Protoc ; 14(6): 1803-1819, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31028374

RESUMO

Bacteria use surface-exposed, proteinaceous fibers called pili for diverse behaviors, including horizontal gene transfer, surface sensing, motility, and pathogenicity. Visualization of these filamentous nanomachines and their activity in live cells has proven challenging, largely due to their small size. Here, we describe a broadly applicable method for labeling and imaging pili and other surface-exposed nanomachines in live cells. This technique uses a combination of genetics and maleimide-based click chemistry in which a cysteine substitution is made in the major pilin subunit for subsequent labeling with thiol-reactive maleimide dyes. Large maleimide-conjugated molecules can also be used to physically interfere with the dynamic activity of filamentous nanomachines. We describe parameters for selecting cysteine substitution positions, optimized labeling conditions for epifluorescence imaging of pilus fibers, and methods for impeding pilus activity. After cysteine knock-in strains have been generated, this protocol can be completed within 30 min to a few hours, depending on the species and the experiment of choice. Visualization of extracellular nanomachines such as pili using this approach can provide a more comprehensive understanding of the role played by these structures in distinct bacterial behaviors.


Assuntos
Caulobacter/ultraestrutura , Fímbrias Bacterianas/ultraestrutura , Corantes Fluorescentes/química , Maleimidas/química , Microscopia de Fluorescência/métodos , Vibrio cholerae/ultraestrutura , Biotina/química , Caulobacter/química , Cisteína/química , Fímbrias Bacterianas/química , Modelos Moleculares , Imagem Óptica/métodos , Coloração e Rotulagem/métodos , Vibrio cholerae/química
14.
Indian J Pathol Microbiol ; 62(2): 274-278, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30971554

RESUMO

Good paraffin sections are key to correct histopathological diagnosis. Xylene is hazardous to health, expensive, and difficult to dispose. Various substitutes have been tried without success. We aimed to examine if 1.7% dishwasher soap (DWS) aqueous solution and refined mineral oil (RMO) for deparaffinization can replace xylene. Fifty tissue blocks consisting of benign and malignant lesions were processed using xylene (A), 1.7% DWS (B), and RMO (C). Each section was evaluated, scored as 0 (inadequate) and 1 (adequate) by two independent pathologists who were blinded to agent used. Following criteria were considered: nuclear staining, cytoplasmic staining, clarity, crispness, and uniformity. Total score of <2 was graded as inadequate for diagnosis and 3-5 as adequate. Statistical analysis was done using the SPSS software by applying chi-square test. Among three methods, B had the best scores in adequacy for cytoplasmic staining (P = 0.001), clarity (P = 0.004), and crispness (P = 0.003). About 1.7% DWS and RMO were found to be effective methods for deparaffinization and can replace xylene.


Assuntos
Coloração e Rotulagem/métodos , Xilenos/química , Carcinoma de Células Escamosas , Vesícula Biliar , Humanos , Óleo Mineral/química , Inclusão em Parafina , Patologia/métodos , Pele , Sabões/química , Estômago
15.
Med Sci (Paris) ; 35(3): 223-231, 2019 Mar.
Artigo em Francês | MEDLINE | ID: mdl-30931906

RESUMO

The proteome is a dynamic system in which protein-protein interactions play a crucial role to model together the cellular phenotype. However, given the inherent limitation of the available technologies to depict the dynamic nature of these interactions, identify protein-protein interaction has for a long time represented an important challenge in proteomic. The recent development of BioID and APEX, two proximity-dependent labeling technologies, opens today new perspectives and yet start changing our vision of protein-protein interaction, and more globally our vision of the proteome. In this review, we describe the recent and conventional tools available to study protein-protein interactions, compare the advantages and limitations of these technics, and discuss the recent progress brought by the proximity-dependent labelling to complete our vision of the proteome, and thus better understand cellular mechanisms.


Assuntos
Mapas de Interação de Proteínas , Proteômica/métodos , Coloração e Rotulagem/métodos , Animais , Biotinilação/métodos , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoma/análise
16.
Prep Biochem Biotechnol ; 49(6): 597-605, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929602

RESUMO

A simple and accurate Nile Red fluorescent method was built to evaluate the lipid content of three different oleaginous yeasts by one standard curve. The staining of cells can be observed clearly by laser scanning confocal microscope, showing that Nile Red can enter into the cells of oleaginous yeasts easily. A series of conditions such as pretreating temperature, cell suspension concentration (OD600), staining time, Nile Red concentration and the type of suspension solvent were learnt systematically to obtain the optimal process parameters for Nile Red staining. After optimization, the fitting curve of Nile Red fluorescent method was established under suitable conditions (pretreating temperature: 50 °C, OD600: 1.0; staining time: 5 mins; Nile Red concentration: 1.0 µg/mL; suspension solvent: PBS) and it had a suitable correlation coefficient (R2 = 0.95) for lipid content measurement of different oleaginous yeasts. By this study, the possibility of lipid content determination of different oleaginous yeasts by one fitting curve can be proven and this will improve the efficiency of researches related to microbial lipid production.


Assuntos
Corantes Fluorescentes/química , Lipídeos/análise , Microscopia Confocal/métodos , Oxazinas/química , Leveduras/química , Cryptococcus/química , Lipomyces/química , Coloração e Rotulagem/métodos , Temperatura Ambiente , Trichosporon/química
17.
Appl Microbiol Biotechnol ; 103(11): 4585-4593, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963206

RESUMO

Beer yeast-modified fluorescent carbon dots were synthesized via a one-step strategy for photoinduced bactericidal functions and bio-imaging in bacterial viability assessment. The proposed carbon dots (CDs) were used as an visible light-triggered antibacterial material, and the antimicrobial activities of the CDs against Gram-negative model bacterial species (Escherichia coli) were evaluated under conditions of varying other experimental parameters including CDs concentrations and treatment times. The result showed that the CDs have excellent antibacterial performance of bactericidal effect within 120 min of under visible-light irradiation. And the bactericidal efficiency increased with the increasing concentration of CDs and visible-light illumination time. Moreover, the CDs with high quantum yield (21%) possess highly negative zeta potential (- 41.7 mV) and low cytotoxicity, the CDs could serve as an efficient dye for bacterial viability evaluation, they could selectively stain dead E. coli rather than live ones, which make dead E. coli be viewed with multicolor fluorescence under different excitation wavelengths.


Assuntos
Antibacterianos/metabolismo , Escherichia coli/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Fármacos Fotossensibilizantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Coloração e Rotulagem/métodos , Cerveja/microbiologia , Carbono/metabolismo
18.
Appl Microbiol Biotechnol ; 103(11): 4443-4453, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989251

RESUMO

The availability of preimmune libraries of antibody fragments allows for the fast generation of binders which can be expressed in both eukaryotic and prokaryotic systems. We exploited the recombinant nature of antibody fragments to demonstrate the possibility of expressing them as functional proteins displayed on the surface of Escherichia coli and by such a way to generate living reagents ready-to-use for diagnostics. Such immunoreagents were effectively exploited without the necessity of any purification step to prepare immunocapture surfaces suitable for the diagnostic of both cancer cells and toxic microalgae. The same nanobody-displaying bacteria were also engineered to coexpress GFP in their cytoplasm. Suspensions of such living fluorescent immunoreagents effectively bound to eukaryotic cells making them visible and quantifiable by flow cytometry analysis and using 96-well plate readers. The collected data showed the suitability of such living immunoreagents for reproducible and inexpensive diagnostic applications.


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Técnicas Citológicas/métodos , Escherichia coli/metabolismo , Proteínas Imobilizadas/metabolismo , Fatores Imunológicos/metabolismo , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/metabolismo , Aderência Bacteriana , Escherichia coli/genética , Proteínas Imobilizadas/genética , Imunoensaio/métodos , Fatores Imunológicos/genética , Proteínas Recombinantes/genética , Anticorpos de Domínio Único/genética , Coloração e Rotulagem/métodos
19.
Nat Commun ; 10(1): 1799, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996301

RESUMO

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Hemaglutininas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Influenza Humana/imunologia , Oligossacarídeos/metabolismo , Animais , Proteínas de Bactérias/genética , Bioensaio/métodos , Células CHO , Cricetulus , Cães , Glicosiltransferases/genética , Voluntários Saudáveis , Helicobacter mustelae/genética , Helicobacter mustelae/metabolismo , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Microscopia Intravital/métodos , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Pulmão/patologia , Células Madin Darby de Rim Canino , Engenharia Metabólica/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Oligossacarídeos/imunologia , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem/métodos
20.
Parasitol Int ; 71: 177-179, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31004805

RESUMO

Giardia cysts stained with hot carbolfuchsin were used as internal controls in a concentration method for surface water samples. The morphological integrity of stained cysts and the stain's stability and intensity were tested with each of the chemical reagents used in the aluminum sulfate flocculation method. No alterations in morphology or color were noted. The stained cyst preparation has a low cost, high stability, and suitability for both light and immunofluorescent microscopy, making it affordable to researchers in low- and middle-income countries.


Assuntos
Corantes/farmacologia , Água Doce/parasitologia , Giardia/isolamento & purificação , Oocistos/isolamento & purificação , Compostos de Alúmen , Fezes/parasitologia , Floculação , Giardia/efeitos dos fármacos , Microscopia de Fluorescência , Oocistos/efeitos dos fármacos , Parasitologia/economia , Parasitologia/métodos , Corantes de Rosanilina/farmacologia , Coloração e Rotulagem/economia , Coloração e Rotulagem/métodos
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